Processing of platelet-containing biological fluids

ABSTRACT

Platelet resuspension solutions, platelet storage solutions and systems for processing platelet-containing biological fluids are disclosed, wherein a platelet resuspension solution comprises an aqueous solution having a pH in the range of from about 4 to about 6: dextrose; and citrate; wherein the solution is substantially free of adenine, and wherein a platelet storage solution comprises an aqueous solution having a pH in the range of from about 6.6 to about 7.8; at least one buffer: dextrose, and citrate; wherein the solution is substantially free of adenine.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 60/479,848, filed Jun. 20, 2003, which is incorporatedby reference.

FIELD OF THE INVENTION

This invention pertains to solutions, methods, and systems forprocessing platelet-containing biological fluids, more particularly, forresuspending and/or storing platelet products.

BACKGROUND OF THE INVENTION

Blood is typically processed to separate various blood components thatcan be separately used. For example, a unit of donated whole blood canbe processed to separate red cells, usually concentrated as packed redcells (PRC), platelets, usually concentrated as platelet concentrate(PC), and plasma. In accordance with typical processing protocols, bloodis processed to form, among other fractions, a platelet-containingfluid, e.g., platelet-rich-plasma (PRP) or buffy coat, that are furtherprocessed (including centrifugation) to form the PC. Moreover, multipleunits of platelets or buffy coat can be pooled before producing thefinal transfusion product.

In accordance with current conventional blood banking practice, PCproduced in a closed system can be stored for up to 5 days before beingused as a transfusion product. In some processing protocols, a plateletadditive solution is added to the platelet-containing fluid (e.g., thebuffy coat) and the platelets are resuspended in the additive solutionbefore the platelets are stored, wherein most of the plasma is removedbefore the additive solution is added. In order to provide optimalplatelet function and viability during storage, it is recommended thatthe platelet-containing fluid (with or without an additive solution) bemaintained at a pH in the range of from 6.8 to 7.4 (European practice),or maintained at a pH of 6.2 or greater (US practice) during the storageperiod. It is also recommended that the platelets be stored in thepresence of glucose to maintain platelet quality.

However, commercially available sterile platelet additive solutions,which have a pH in the range of 7.0-7.2, do not contain glucose, due todifficulties (e.g., glucose caramelization) encountered when steamsterilizing glucose-containing additive solutions having such a pHrange. Accordingly, since plasma contains glucose, when these sterilizedcommercially available solutions are used, at least about 10% of theinitial volume of plasma from a whole blood unit must remain with theplatelets (e.g., about 30-50 ml of plasma remains from the buffy coat,or about 40-75 ml of plasma remains from the PRP, the rest of the plasmais removed) to provide sufficient glucose for the storage period. Inview of the volume of additive solution added to the platelet-containingfluid, the platelets are diluted, and this can provide an increasedfluid load on a patient receiving the platelets as a transfusionproduct.

Additionally, platelets may become activated during the processing ofblood to concentrate the platelets (including during the subsequentresuspension of the platelets in the additive solution), leading toplatelet aggregation and a loss of viable platelets in the transfusionproduct.

The present invention provides for ameliorating at least some of thedisadvantages of the prior art. These and other advantages of thepresent invention will be apparent from the description as set forthbelow.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention provides a platelet resuspensionsolution, comprising an aqueous solution having a pH in the range offrom about 4 to about 6, dextrose (glucose), and citrate, wherein thesolution is essentially free of adenine. In a preferred embodiment, theplatelet resuspension solution comprises a sterilizable solution.

In another embodiment, the invention provides a platelet storagesolution, comprising an aqueous solution having a pH in the range offrom about 6.6 to about 7.8, dextrose, citrate, and a buffer, whereinthe solution is essentially free of adenine. In a preferred embodiment,the buffer comprises sodium bicarbonate.

A platelet processing system according to an embodiment of the inventioncomprises a flexible bag and platelet resuspension solution contained inthe bag.

In yet another embodiment, a platelet storage system is provided,comprising a flexible bag suitable for containing a platelet-containingsolution, the bag having gas permeable side walls, and furthercomprising a buffering material. The buffering material can be in thebag, or in a compartment communicating with the bag.

In another embodiment, a biological fluid processing system is provided,comprising the platelet processing system, and at least one, andpreferably, at least two, additional flexible bags. In a more preferredembodiment, at least one of the additional flexible bags comprises a gaspermeable bag.

Methods of using the platelet additive solutions, the plateletprocessing system, and the platelet storage system, and the biologicalfluid processing systems are also provided.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a platelet resuspensionsolution, comprising an aqueous solution having a pH in the range offrom about 4 to about 6, preferably in the range from about 5 to about5.5, dextrose (glucose), and citrate, wherein the solution issubstantially free of adenine. In a preferred embodiment, the plateletresuspension solution comprises a sterilizable solution.

In another embodiment, the invention provides a platelet storagesolution, comprising an aqueous solution having a pH in the range offrom about 6.6 to about 7.8, preferably in the range of from about 6.8to about 7.4, dextrose, citrate, and a buffer, wherein the solution isessentially free of adenine. In a preferred embodiment, the buffercomprises sodium bicarbonate.

Preferred embodiments of the resuspension and/or storage solutionsinclude acetate, more preferably, sodium acetate. Without being limitedto any particular mechanism, it is believed the presence of sodiumacetate in the resuspension solution and/or storage solution reducesglycolysis, thus making it easier to control of pH of the solution(s).With respect to the storage solution, this may reduce the amount ofbuffer utilized in the solution.

Typically, the platelet resuspension and/or platelet storage solutionalso includes electrolytes for ionic balance. For example, preferredembodiments of the solution(s) include at least one of, and preferably,at least two of, sodium chloride (for example, physiological salinesolution), potassium chloride, and magnesium chloride, as theseelectrolytes are more natural to the human body (e.g., as they are moresimilar to those electrolytes found in plasma).

The platelet resuspension and/or platelet storage solution can alsoinclude additional components such as, for example, phosphate and/orcitric acid.

Typically, the platelet resuspension and/or platelet storage solutionhas an osmolarity in the range of from about 260 to about 380 mOsmAL,preferably, in the range of from about 280 to about 320 mOsm/L, asmeasured in accordance with U.S. Pharmacopeia (USP) 24-NF19.

In accordance with an embodiment of a platelet resuspension solutionaccording to the invention, the solution has a pH in the range of fromabout 4 to about 6, preferably, in the range of from about 4.6 to about5.7, even more preferably, in the range from about 5 to about 5.7. Inpreferred embodiment, the platelet resuspension solution is asterilizable solution, more preferably, a steam sterilizable solution.

In accordance with an embodiment of a platelet storage solutionaccording to the invention, the solution has a pH in the range of fromabout 6.6 to about 7.8, preferably, in the range of from about 6.8 toabout 7.4. Typically, at a temperature in the range of from about 22° C.to about 24° C., the solution has a pH in the range of from about 6.8 toabout 7.6, and at a temperature in the range of from about 27° C. toabout 39° C., the solution typically has a pH in the range of from about7 to about 7.8.

Typical embodiments of platelet resuspension and/or platelet storagesolutions include concentrations in the range of from about 10 to about45 mM glucose, about 6 to about 10 mM sodium citrate, and 0 to about 25mM sodium acetate. In those embodiments also including electrolytes, thesolution(s) typically include at least one, and more preferably, each ofthe following, in the following concentration(s): in the range of fromabout 3 to about 7 mM potassium chloride, about 1 to about 5 mMmagnesium chloride, and about 70 to about 130 mM sodium chloride. Thesolution(s) may also include, for example, in the range of from 0 toabout 5 mM citric acid and/or in the range of about 3.5 to about 7 mMsodium phosphate (in some embodiments, in the range of about 3.5 toabout 4.5 mM sodium phosphate).

Preferred embodiments of platelet resuspension and/or platelet storagesolutions include concentrations in the range of from about 15 to about30 mM glucose, about 7 to about 9 mM sodium citrate, and 8 to about 18mM sodium acetate. In those embodiments also including electrolytes, thesolution(s) typically include at least one, and more preferably, each ofthe following in the following concentration(s): in the range of fromabout 3 to about 5 mM potassium chloride, about 2 to about 4 mMmagnesium chloride, and about 80 to about 120 mM sodium chloride. Thesolution(s) may also include, for example, citric acid having aconcentration in the range of from 1 to about 4 mM and/or sodiumphosphate having a concentration in the range of about 3 to about 6.5mM.

In those embodiments wherein the platelet resuspension solution includescitric acid and sodium citrate, the mM ratio of citric acid/sodiumcitrate is preferably in the range of from about 0.1 to about 0.5.

With respect to a buffer, e.g., as included in the platelet storagesolution, a preferred buffer is sodium bicarbonate, in a concentrationin the range of from about 5 to about 20 mM, more preferably in therange of from about 8 to about 15 mM. In accordance with preferredembodiments of the invention, the buffering material is sterilizedbefore it is utilized in forming an embodiment of the platelet storagesolution or before it is added to a platelet-containing resuspensionsolution.

As used herein, a solution substantially free of adenine has an adenineconcentration of 0.2 mM or less, preferably, 0.1 MM or less.

A platelet processing system according to an embodiment of the inventioncomprises a flexible bag and the platelet resuspension solution asdescribed above contained in the bag. In some embodiments, the flexiblebag is suitable for containing a platelet- and plateletresuspension-containing solution

In yet another embodiment, a platelet storage system is provided,comprising a flexible bag suitable for containing (preferably, forstoring) a platelet-containing solution, the bag being a gas permeablebag, and further comprising a buffering material. In some embodiments ofthe system, the bag contains the buffering material, or the systemfurther comprises a compartment communicating with the flexible bag,wherein the compartment, preferably a squeezable compartment, containsthe buffering material. In an embodiment of the system, the bag containsthe platelet storage solution as described above therein.

In another embodiment, a biological fluid processing system is provided,comprising the platelet processing system as described above, and atleast one, and preferably, at least two, additional flexible bags. In amore preferred embodiment, at least one of the additional flexible bagscomprises a gas permeable bag.

In another embodiment, a biological fluid processing system comprisesthe platelet processing system as described above, and an apheresisbowl, chamber, or tube, suitable for containing a platelet-containingbiological fluid. The system can include at least one additionalcontainer, e.g., a flexible, gas permeable bag.

In accordance with the invention, platelets can be effectivelyresuspended in the platelet resuspension solution (in some embodiments,resuspended in the platelet storage solution) while reducing activationand the formation of aggregates, thus increasing the yield of valuableplatelets for transfusion. Moreover, the platelets can be stored whilemaintaining high platelet quality, thus providing a high yield ofuseable platelets for transfusion.

Furthermore, the platelets can be prepared and stored while requiringless plasma in the stored product than is conventionally used, thusincreasing the yield for valuable plasma for other uses. For example,while conventional methods require utilizing about at least about 10% ofthe initial volume of plasma in preparing platelet products for storage,platelet products suitable for storage can be prepared in accordancewith embodiments of the invention utilizing about 50% or less plasmathan that used in the conventional methods, typically, about 40% orless, and in some embodiments, about 30% or less.

For example, in a conventional method wherein about 30 to about 35 ml ofplasma from buffy coat is used for preparing a platelet product forstorage, embodiments of the invention can utilize about 10 to about 15ml of plasma. In another conventional method wherein about 50 to about60 ml of plasma from platelet-rich-plasma (PRP) is used for preparing aplatelet product for storage, embodiments of the invention can utilizeabout 5 to about 15 ml of plasma

There are other advantages associated with the use of less plasma Forexample, platelet products can be prepared having less total volume,thus decreasing the load on the patient receiving the transfusionproduct. Moreover, the platelet products can be prepared containing agreater number of platelets without requiring a great increase in thetotal volume of the product to obtain the additional platelets. Yetanother advantage is the additive solution according to the inventionadds less fluid volume to the platelet product than conventionaladditives. For example, while conventional additives would add about 300ml of additive solution to, for example, 4 pooled buffy coats, preferredembodiments of the invention would add about 200 ml of the inventiveadditive solution or less. With respect to non-pooled platelet products,e.g., single random donor units, units can be prepared having, ifdesired, a total volume of about 40 to about 50 ml.

Typically, the platelet resuspension solution and/or platelet storagesolution is essentially free of photoactive agents (e.g.,photosensitizers for use in inactivating microorganisms). However, thesolution(s) can be used with photosensitizers, and, in microorganisminactivation protocols such as viral inactivation protocols, thepresence of less plasma allows less inactivation agent (e.g., psoralenssuch as methylene blue), or a lower dose of the inactivation agent(e.g., UV light) to be utilized during the protocol. Thus, there can beless inactivation agent to be subsequently removed and/or plateletdamage can be reduced.

In accordance with preferred embodiments of a method according to theinvention, a platelet-containing biological fluid (e.g., comprisingapheresis platelets, platelets obtained from platelet-rich-plasma orplatelets obtained from pooled buffy coats), is combined with a plateletresuspension solution to form a platelet-containing plateletresuspension solution, and the platelets are resuspended in thesolution. In some embodiments, e.g., some apheresis systems, theplatelet resuspension solution comprises an elutriation solution. Theplatelet-containing platelet resuspension solution is further processed,which includes adding a buffer, and the buffered platelet-containingfluid is stored in a container (preferably a gas permeable flexible bag)for a desired period of time before further use, e.g., as a transfusionproduct that is administered to a patient.

Embodiments of the method can include pooling two or more volumes ofplatelet-containing fluid (e.g., two or more units of buffy coat), andmixing pooled platelet-containing fluid with a platelet-resuspensionsolution to provide a pooled platelet-containing platelet resuspensionsolution. For example, in one embodiment, 4-6 units of whole blood areeach processed to provide sedimented red cells, buffy coat, andplatelet-poor-plasma (PPP), and the components are separated. Asexplained above, in preferred embodiments, a greater volume of PPP canbe removed when separating the PPP from the buffy coat, as the PPP isnot needed to provide glucose. The buffy coats (the platelet-containingfluid) from each unit of whole blood are pooled, the pooledplatelet-containing fluid is mixed with a platelet resuspensionsolution, and the platelets are re-suspended in the resuspensionsolution. The fluid is further processed so that platelets can beprepared for storage. For example, further processing includes adding abuffering material (typically after centrifugation and separation ofbiological fluid components), and the resultant supernatant pooledplatelet-containing fluid can subsequently be separated from thesedimented red and white blood cells. The separated pooledplatelet-containing fluid (containing the buffered resuspensionsolution, thus forming an embodiment of the. storage solution), can bestored as described above before further use.

In one embodiment that does not include pooling, a unit of whole bloodis centrifuged to form supernatant platelet-rich-plasma (PRP) andsedimented packed red blood cells, and the PRP is expressed to, forexample, a container such as a blood bag, e.g., a plasticized satellitebag. The PRP is subsequently centrifuged to concentrate the platelets atthe bottom of the bag, and the supernatant plasma is expressed from thebag. As explained above, in preferred embodiments, a greater volume ofplasma can be removed when separating the plasma from the concentratedplatelets, as this plasma is not needed to provide glucose. After theplasma is removed, platelet resuspension solution (e.g., about 40 toabout 50 ml) is added to the bag, and the platelets are resuspended inthe resuspension solution. In some embodiments, the platelets areresuspended in the resuspension solution after the platelets have restedunagitated for a period of time, for example, about 1-2 hours.Subsequently, a buffer is added, thus forming an embodiment of thestorage solution, and the buffered, platelet-containing solution can bestored as described above before further use.

In another embodiment, after the PRP is centrifuged and the supernatantplasma is removed as described above, non-platelet containing bufferedplatelet storage solution (e.g., about 40 to about 50 ml of thesolution) is added to the bag, and the platelets are (in someembodiments, after the platelets have rested unagitated for a period oftime) resuspended in the buffered storage solution, and can be stored inthe solution.

In accordance with another embodiment that does not include pooling,e.g., wherein the biological fluid is processed in an apheresis system,concentrated platelets are prepared using the apheresis system (e.g.,including, but not limited to, an apheresis system such as a BaxterFenwall Amicus® Separator or a Baxter Fenwall CS 3000 plus) in acontainer (e.g., a collection chamber), and an embodiment of theresuspension solution (e.g., about 100 to about 300 ml for singleproduct platelets) is added to the concentrated platelet fluid,preferably, without adding plasma to the concentrated platelet fluid.The platelets are resuspended in the resuspension solution (in someembodiments, the platelets are resuspended after the platelets haverested unagitated for a period of time). Subsequently, a buffer isadded, thus forming the storage solution, and the buffered,platelet-containing solution can be stored as described above beforefurther use.

In another embodiment, after concentrated platelets are prepared usingthe apheresis system in a container (e.g., a collection chamber) asdescribed above, an embodiment of the non-platelet containing bufferedstorage solution (e.g., about 100 to about 300 ml of the solution forsingle product platelets) is added to the concentrated platelet fluid,preferably, without adding plasma to the concentrated platelet fluid.The platelets are (in some embodiments, after the platelets have restedunagitated for a period of time) resuspended in the buffered storagesolution, and can be stored in the solution.

In preferred embodiments of the invention, leukocytes are depleted fromthe platelets. For example, a platelet-containing fluid, that can beprepared, for example, in an apheresis system or from a donated unit ofwhole blood, e.g., PRP, buffy coat, or the supernatant layer comprisingplatelets formed after pooled buffy coats are centrifuged, can be passedthrough a leukocyte depletion filter (preferably a leukocyte depletionfilter including a leukocyte depletion- and red cell barrier-medium) toprovide leukocyte-depleted platelets. A platelet-containing fluid can beleukocyte-depleted before forming the platelet-containing plateletresuspension solution, or before forming a platelet-containing storagesolution, or after forming the platelet-containing platelet resuspensionsolution, but before adding a buffer. Preferably, a platelet-containingfluid is filtered before a buffer is added to the fluid.

In those embodiments wherein a platelet-containing fluid is combinedwith a buffered platelet storage solution (e.g., some embodimentswherein PRP is centrifuged and the supernatant plasma is removed, andthe buffered platelet storage solution is mixed with the concentratedplatelets), platelets are preferably filtered before aplatelet-containing fluid is combined with a buffered platelet storagesolution.

A typical embodiment of the method further comprises storing thebuffered platelet-containing solution (preferably, wherein the plateletshave been leukocyte-depleted) in the container for at least 2 days,preferably, at least 5 days, and in some embodiments, at least 7 days.Preferably, the pH of the fluid is maintained within the range of about6.8 to about 7.4 during the storage period, more preferably, wherein thefluid is stored in a gas permeable flexible bag.

If desired, a preferred embodiment of the method further comprisesadministering platelets to a patient.

In accordance with current U.S. practice, platelet-containing biologicalfluids prepared in closed systems (with or without additive solutions)can be stored for 5 days before use, e.g., as transfusion products, andplatelets processed according to the invention can be stored for thatperiod of time. However, platelets produced in accordance withembodiments of the invention can remain viable for longer periods oftime, e.g., they remain viable after 7 days of storage, after 10 days ofstorage, and even after 14 days of storage. Accordingly, should theregulations in the U.S., or any other country be changed, embodiments ofthe invention allow for platelet storage for longer than 5 days, e.g.,up to about 7 days or more, or 10 days, or even, 14 days, or more.

The viability of the platelets can be determined by a variety of methodsknown in the art. Typically, in determining viability, at least one, andmore preferably, two or more, of the following are evaluated: plateletcount, pH, pO₂, pCO₂, bicarbonate, streaming (or swirling), hypotonicshock response (% HSR), extent of shape change (% ESC), % discs(platelet morphology), CD62 level (p-selectin), plasma glucose, plasmalactate, ATP level, and in vivo radiolabeling studies.

A variety of containers, preferably flexible blood bags, are suitablefor use in accordance with the invention. The containers should besterilizable in accordance with conventional protocols, e.g., at leastone of steam sterilization, gamma sterilization, and ethylene oxidesterilization.

In some embodiments, the platelets can be resuspended in the plateletresuspension solution in one container (for example, in the collectionchamber of an apheresis system, or in a flexible blood bag), and thebuffered platelet-containing solution can be formed in anothercontainer, for example. Typically, the container utilized for storingthe platelets (that may be the container in which the buffered plateletstorage solution is formed) is a gas permeable container, e.g., acontainer allowing suitable gas transmission into and/or out of theinterior volume of the container.

Examples of suitable flexible containers, wherein the containers are gaspermeable, include, but are not limited to, polyolefin elastomer bags asdescribed in International Publication No. WO 02065976, bags preparedfrom a film comprising ultra high molecular weight plasticized PVC asdescribed in U.S. Pat. No. 5,721,024, and bags prepared from a filmcomprising plasticized PVC as described in U.S. Pat. No. 4,280,497.

Preferably, the polymeric film used in manufacturing gas permeable bagshas a 22° C. room air oxygen transmission of about 12 μmoles or greaterO₂/hr/350 cm² film surface area. In some embodiments, the 22° C. roomair oxygen transmission is 15 μmoles or greater O₂/hr/350 cm² filmsurface area, preferably, about 18 μmoles or greater O₂/hr/350 cm² filmsurface area, and even more preferably, about 20 μmoles or greaterO₂/hr/350 cm² film surface area.

The containers used in accordance with some embodiments are alsoresilient to temperature fluctuations, e.g., they can withstand lowtemperatures during freezing, e.g., when processing plasma.

In some embodiments, at least one container, e.g., wherein the containercomprises a gas permeable container, is free of, or essentially free of,plasticizers such as di (2-ethylhexyl) phthalate (DEHP), tri(2-ethylhexyl) trimellitate (TOTM), and citrate ester plasticizers suchas n-butryl tri-n-hexyl citrate (BTHC). However, one or more containers(e.g., the polymeric film) can include modifiers and/or additives suchas, for example, at least one of an antistatic, antiblock, a stabilizer,and antioxidant, e.g., for use in processing the film for making thecontainers.

As described in International Publication No. WO 02065976, a resin isused in producing the polymeric film used in manufacturing a polyolefinelastomer bag and the resin comprises at least one copolymer comprisingethylene and an acrylate, preferably comprising ethylene and an alkylacrylate. The resin can comprise a plurality of copolymers, e.g., ablend comprising a first copolymer comprising ethylene and a first alkylacrylate, and a second copolymer comprising ethylene and a second alkylacrylate.

In some embodiments, the copolymer comprises ethylene and at least about20 weight percent alkyl acrylate based upon the combined weight of theethylene and the alkyl acrylate. For example, the copolymer can compriseethylene and at least about 22 weight percent alkyl acrylate, orethylene and at least about 24 weight percent alkyl acrylate. The term“alkyl” herein refers to an alkyl group having from 1 to about 10 carbonatoms, preferably from 1 to about 6 carbon atoms, and more preferablyfrom 1 to about 4 carbon atoms. In even more preferred embodiments, thealkyl acrylate is methyl acrylate or butyl acrylate. For example, theresin can comprise a copolymer comprising ethylene, and at least about20 wt. % methyl acrylate or at least about 20 wt. % butyl acrylate. Inother embodiments, the resin comprises a copolymer comprising ethylene,and at least about 22 wt. % methyl acrylate or at least about 22 wt. %butyl acrylate, or ethylene and at least about 24 wt. % methyl acrylateor at least about 24 wt. % butyl acrylate.

Suitable resins include, for example, resins commercially availablefrom, for example, Eastman Chemical Company (Kingsport, Tenn.), AtofinaChemicals, Inc. (Philadelphia, Pa.) and Dupont (Wilmington, Del.). Forexample, a variety of resins commercially available from EastmnanChemical Company referred to as EMAC® (including EMAC+®), EBAC®(including EBAC+®), and EMAC/EBAC® are suitable. Illustrative examplesof such resins are ethylene butyl acrylate copolymer (EBAC) resin, e.g.,EBAC SP1802 and SP1903 specialty copolymers, and ethylene methylacrylate copolymer (EMAC) resin, e.g., EMAC SP1305, SP1307, SP1330,SP1400, SP2202, SP2207, SP2220, SP2260 and SP2268, specialty copolymers.Illustrative suitable resins commercially available from AtofinaChemicals, Inc., include, for example, those resins referred to asLOTRYL™ resins (e.g., LOTRYL™ EBA and LOTRYL™ EMA) and illustrativesuitable resins commercially available from DuPont include, for example,those resins referred to as ELVALOYT™ resins (e.g., ELALOY™ AC).

As described in U.S. Pat. No. 5,721,024, a flexible container cancomprise a polyvinyl chloride (PVC) film manufactured from a polyvinylchloride compound, said compound comprising an ultra high molecularweight polyvinyl chloride resin having an inherent viscosity rangingfrom about 1.25 to about 2.00, as measured by ASTM D-1243; and about 43weight percent or more (typically, in the range of from about 43 toabout 57 weight percent) of a plasticizer. Preferably, the plasticizeris one from the group of plasticizers consisting of: tri (2-ethylhexyl)trimellitate; di-(2-ethylhexyl) phthalate; acetyl tri-n-butyl citrate;n-butyryl tri-n-hexyl citrate; acetyl tri-n-octyl citrate; and acetyltri-n-decyl citrate.

The bags according to the invention can have any suitable size, shape,internal volume and/or thickness. The bags can be made from thepolymeric film and resin described herein using conventional techniquesknown and used in the industry. Illustratively, the bag can be arrangedfrom a single sheet of sheet of film (e.g., folded over at the end wherethe ports are arranged and sealed around the other edges), two sheets offilm, from a collapsed blown bubble of film (sometimes referred to as“lay flat tubing”), and the like. The bags are typically extruded, butcan be blow molded or formed by other appropriate methods known in theart.

The preferred wall thickness of gas permeable containers for storingplatelet-containing fluids using the polymeric film can be in theconventional range ofqabout 0.005 to about 0.025 inch (about 0.13 toabout 0.64 mm), preferably about 0.010 inch to about 0.018 inch (about0.25 to about 0.46 mm), with about 0.012 to about 0.015 inch (about 0.30to about 0.38 mm) being most preferred. This wall thickness results incontainers having sufficient tensile strength to withstand conventionaluse in the collection and processing of blood and blood components.

In typical embodiments of gas permeable containers according to theinvention, each side wall is a single layer of film.

In a preferred embodiment, the gas permeable bag is configured to allowat least one material (e.g., a dry material, or a liquid), morepreferably, a buffering material, even more preferably a buffer that canbe subjected to sterilization conditions without substantialdegradation, to be mixed with a fluid, e.g., the platelet-containingplatelet resuspension solution mixture, or a non-platelet containingresuspension solution, in the bag. The material can be present (e.g., asa tablet) in the interior volume of the bag, e.g., so that theplatelet-containing platelet resuspension solution mixture contacts thematerial when the solution mixture is passed into the bag.

Alternatively, gas permeable container can include at least twocompartments, e.g., a larger compartment for containing aplatelet-containing platelet resuspension solution mixture, and at leastone smaller compartment containing the material wherein the bag isarranged to allow the material in the smaller compartment to be passedto the larger compartment. In yet another embodiment, an additionalcontainer is arranged so that material from the additional container canbe passed into the container containing the platelet-containing plateletresuspension solution mixture. Typically, the smaller compartment or theadditional container is adapted to contain a buffer. Between the smallercompartment and the larger compartment, and between the additionalcontainer and the gas permeable container, is a closure means,preferably an externally manipulable closure means (in some embodiments,an in-line frangible valve), that allows the buffer to be mixed with theplatelet-containing platelet resuspension solution mixture when desired.In one embodiment, the gas permeable bag includes at least twocompartments as disclosed in U.S. Pat. Nos. 4,902,287 and 4,994,057,wherein at least one smaller compartment is squeezable, i.e., thesmaller compartment comprises a resilient material that causes thecompartment to generally return to its original shape after it has beendeformed by external pressure on the compartment walls.

While a variety of buffers are suitable for use in accordance with theinvention, a preferred material is sodium bicarbonate. The bufferingmaterial is preferably in dry form, e.g., in powder or tablet form. Asnoted above, preferably, the buffer can be subjected to sterilizationconditions without substantial degradation.

A variety of leukocyte depletion filters comprising leukocyte media aresuitable for use in according to the invention. Alternatively, oradditionally, in some embodiments, the leukocyte depletion filtercomprises a red cell barrier medium, or a combined red cell barrierleukocyte depletion medium. Suitable filters and media include, but arenot limited to, those disclosed in U.S. Pat. Nos. 4,880,548, 5,100,564,5,152,905, 5,472,621 and 5,670,060.

The following definitions are used in accordance with the invention.

Biological Fluid. A biological fluid includes any treated or untreatedfluid associated with living organisms, particularly blood, includingwhole blood, warm or cold blood, and stored or fresh blood; treatedblood, such as-blood diluted with at least one physiological solution,including but not limited to saline, nutrient, and/or anticoagulantsolutions; blood components, such as platelet concentrate (PC),platelet-rich plasma (PRP), platelet-poor plasma (PPP), platelet-freeplasma, plasma, fresh frozen plasma (FFP), components obtained fromplasma, packed red cells (PRC), transition zone material or buffy coat(BC); blood products derived from blood or a blood component or derivedfrom bone marrow; stem cells; red cells separated from plasma andresuspended in physiological fluid or a cryoprotective fluid; andplatelets separated from plasma and resuspended in physiological fluidor a cryoprotective fluid. The biological fluid may have been treated toremove some of the leukocytes before being processed according to theinvention. As used herein, blood product or biological fluid refers tothe components described above, and to similar blood products orbiological fluids obtained by other means and with similar properties.

A “unit” is the quantity of biological fluid from a donor or derivedfrom one unit of whole blood. It may also refer to the quantity drawnduring a single donation. Typically, the volume of a unit varies, theamount differing from patient to patient and from donation to donation.Multiple units of some blood components, particularly platelets andbuffy coat, may be pooled or combined, typically by combining four ormore units.

As used herein, the term “closed” refers to a system that allows thecollection and processing (and, if desired, the manipulation, e.g.,separation of portions, separation into components, filtration, storage,and preservation) of biological fluid, e.g., donor blood, blood samples,and/or blood components, without the need to compromise the sterileintegrity of the system. A closed system can be as originally made, orresult from the connection of system components using what are known as“sterile docking” devices. Illustrative sterile docking devices aredisclosed in U.S. Pat. Nos. 4,507,119, 4,737,214, and 4,913,756.

Sterilizable preferably means the capability of being subjected totemperatures of at least about 114° C. for at least about 30 minutes (orexposed to at least about 2.5 megarads of gamma radiation) withoutsignificant degradation of a given product. In the case of dry or liquidcompounds, chemicals, or components, such compounds, chemicals andcomponents preferably retain at least about 75% by weight of theirinitial pre-sterilization chemical identity and utility after havingbeen subjected to the above sterilization conditions.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example describes the preparation of an embodiment of the plateletresuspension solution, as well as the preparation of an embodiment of aplatelet processing system.

A 200 mL platelet resuspension solution with a pH of 5.3 and acalculated osmolarity of 340 mOsm/L is prepared having the following:g/L mM Dextrose H₂O 5.94 30 Potassium Chloride 0.37 5 Magnesium Chloride6H₂O 0.61 3 Sodium Chloride 6.43 110 Citric acid 0.48 2.5 Sodium Citrate2H₂0 2.21 7.5 Sodium Phosphate 0.55 4 monobasic H₂O Sodium Acetate 3H₂O2.04 15

The solution is placed in a plasticized PVC bag, and steam sterilized toprovide a platelet processing system.

EXAMPLE 2

This example described the preparation of an embodiment of a plateletstorage system according to the invention.

A gas permeable polyolefin elastomer platelet storage bag is prepared asgenerally described in International Publication No. WO 02065976. Thebag contains a buffering material, a sodium bicarbonate tablet (168 mg).

The bag, including the tablet, is gamma sterilized.

EXAMPLE 3

This example describes the resuspension of a platelet-containingsolution using embodiments of a platelet storage solution, a plateletprocessing system, and a platelet storage system, according to theinvention.

Four units of anticoagulated whole blood are each processed intop/bottom bags to produce supernatant platelet-poor-plasma (PPP), buffycoat (BC), and sedimented packed red blood cells (PRC), wherein the PPPand PRC are separated from the BC. In separating the PPP from the BC,about 10-15 mL of PPP remains with the BC.

The four top/bottom bags each containing BC therein are sterileconnected together in series. The platelet processing system asdescribed in Example 1, containing the 200 mL of resuspension solution,is sterile connected to the top port of the first (top) top/bottom bagin the series. A second plasticized PVC bag is sterile connected to thebottom port of the last (bottom) top/bottom bag in the series.

A filter including a leukocyte depletion and red cell barrier medium asdescribed in U.S. Pat. No. 5,670,060 is obtained, and sterile connectedbetween the second plasticized PVC bag and the platelet storage systemdescribed in Example 2.

The four units of BC connected in series are drained from each bag andpooled into the plasticized PVC bag, and the sterile resuspensionsolution is passed through the top port of the first bag and through theother 3 top/bottom bags into the PVC bag.

The conduit between the bottom port of the last top/bottom bag and thesecond PVC bag is heat-sealed and cut.

The platelet- and platelet resuspension solution-containing fluid iscentrifuged to provide a supernatant layer including platelets, and asediment layer including red blood cells.

EXAMPLE 4

This example describes the leukocyte-depletion and storage of aplatelet-containing solution using an embodiment of a platelet storagesystem, according to the invention.

The supernatant layer described in Example 3 is passed from the secondPVC bag, through the filter, into the platelet storage system, whereinthe supernatant layer contacts the sodium bicarbonate tablet in thepolyolefin elastomer bag. The concentration of sodium bicarbonate in thesolution is 10 mM.

The conduit between the outlet of the filter and the port of theplatelet storage system is heat-sealed and cut.

The gas permeable bag including the buffered solution therein is placedon a horizontal shaker in a 20-24° C. controlled environmental chamber.Platelet quality is measured on days 2, 5, 7, and 9 of storage.

Additionally, the platelet concentration in the container is determinedto be greater than 1.4 ×10⁹ platelets/mL.

The pH is well maintained at a pH of 7.2 to 7.4 during the storageperiod, and the platelets remain viable as reflected by analysis of theektent of shape change (ESC) and the hypotonic shock response (HSR).

Examples 3 and 4 show platelets can be resuspended and stored inaccordance with embodiments of the invention, and platelet viability (ata concentration of over 1.4×10⁹ platelets/mL) can be maintained for atleast a 9 day storage period.

EXAMPLE 5

This example describes the preparation of an embodiment of the plateletstorage solution.

A 50 mL solution with a pH of 5.3 and a calculated osmolarity of 340mOsm/L is prepared having the following: g/L mM Dextrose H₂O 5.94 30Potassium Chloride 0.37 5 Magnesium Chloride 6H₂O 0.61 3 Sodium Chloride6.43 110 Citric acid 0.48 2.5 Sodium Citrate 2H₂0 2.21 7.5 SodiumPhosphate 0.55 4 monobasic H₂O Sodium Acetate 3H₂O 2.04 15

A plasticized PVC bag is obtained, and a squeezable compartment isformed from plasticized PVC and attached to the bag via a conduit asgenerally described in U.S. Pat. No. 4,902,287. The squeezablecompartment includes sodium bicarbonate powder (168 mg). The conduitincludes an in-line frangible valve. The plasticized PVC bag is alsopart of a closed multiple bag blood processing set including acollection bag, a plurality of satellite bags, and a filter comprising aleukocyte depletion and red cell barrier medium as described in U.S.Pat. No. 5,152,905.

The solution is placed in the plasticized PVC bag, and the solution, aswell as the sodium bicarbonate in the squeezable container, are steamsterilized.

The in-line frangible valve is manipulated to open the flow path in theconduit between the PVC bag and the squeezable container. The PVC bag isinverted, and the squeezable compartment is squeezed several times topass buffer into the bag and draw liquid into the compartment.

The resultant buffered platelet storage solution has a pH of 7.3, and asodium bicarbonate concentration of 10 mM.

EXAMPLE 6

This example describes the resuspension of a platelet-containingsolution in an embodiment of the buffered platelet storage solutionaccording to the invention.

A unit of anticoagulated whole blood is processed in a plasticizedcollection bag (which is one of the bags in the multiple bag setdescribed in Example 5) to provide a supernatant layer comprisingplatelet-rich-plasma (PRP) and a sediment layer comprising packed redblood cells. The PRP is passed through the filter comprising a leukocytedepletion and red cell barrier medium into a gas permeable plasticizedPVC satellite bag (wherein the gas permeable bag is described in U.S.Pat. No. 4,280,497) to provide leukocyte-depleted PRP in the satellitebag.

The leukocyte-depleted PRP is subjected to hard spin centrifugation, toprovide concentrated platelets in the bottom of the satellite bag, andplatelet-poor-plasma (PPP). About 250 ml of PPP is expressed from thesatellite bag, leaving about 5 ml of plasma in the bag with theconcentrated platelets. The 50 ml of buffered platelet storage is passedfrom the plasticized PVC bag described in Example 5 into the satellitebag, and, after a rest period of about 1.5 hours, the platelets areresuspended in the platelet storage solution wherein the gas permeablesatellite bag is placed on a horizontal shaker in a 20-24 ° C.controlled environmental chamber.

Platelet quality is measured on days 2, 5, 7, and 9 of storage.

The pH is well maintained at a pH of 7.2 to 7.4 during the storageperiod, and the platelets remain viable as reflected by analysis of theextent of shape change (ESC) and the hypotonic shock response (HSR).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors, for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A platelet resuspension solution comprising: an aqueous solutionhaving a pH in the range of from about 4 to about 6; dextrose; andcitrate; wherein the solution is substantially free of adenine.
 2. Thesolution of claim 1, further comprising sodium acetate.
 3. The solutionof claim 1, comprising a sterilizable solution.
 4. The solution of claim1, comprising a steam sterilizable solution.
 5. The solution of claim 2,further comprising at least one electrolyte selected from potassiumchloride, magnesium chloride, and sodium chloride.
 6. The solution ofclaim 1, further comprising citric acid and/or sodium phosphate.
 7. Thesolution of claim 1, having a pH in the range of from about 5 to about5.7.
 8. (canceled)
 9. The system of claim 24, wherein the solutionfurther comprises at least one electrolyte selected from potassiumchloride, magnesium chloride, and sodium chloride.
 10. The system ofclaim 24, wherein the solution further comprises citric acid and/orsodium phosphate.
 11. The system of claim 24, wherein the solutionfurther comprises having a pH in the range of from about 6.8 to about7.4.
 12. The system of claim 24, wherein the solution further comprisesat least one buffer comprises sodium bicarbonate.
 13. The system ofclaim 24, wherein the solution further comprises platelets containedtherein.
 14. A platelet processing system comprising: a flexible bag;and, an amount of the platelet resuspension solution of claim 1,contained in the bag.
 15. The system of claim 14, comprising at leastabout 50 cc of platelet resuspension solution.
 16. The system of claim14, comprising at least about 200 cc of platelet resuspension solution.17. The system of claim 14, comprising a sterilizable system.
 18. Thesystem of claim 17, comprising a steam sterilizable system.
 19. Thesystem of claim 14, further comprising a buffering material, separatedfrom the platelet resuspension solution.
 20. The system of claim 19,wherein the buffering material comprises sodium bicarbonate.
 21. Thesystem of claim 20, wherein the sodium bicarbonate is in solid form. 22.The system of claim 21, wherein the sodium bicarbonate is in the bag.23. The system of claim 19, comprising a sterilized buffering material.24. A platelet storage system comprising: a gas permeable flexible bag;an amount of the platelet storage solution contained in the bag, thesolution comprising an aqueous solution having a pH in the range of fromabout 6.6 to about 7.8; at least one buffer: dextrose; and citrate;wherein the solution is substantially free of adenine, and a leukocytedepletion filter assembly in fluid communication with the bag.
 25. Thesystem of claim 24, comprising at least about 50 cc of platelet storagesolution.
 26. The system of claim 24, comprising at least about 200 ccof platelet storage solution.
 27. The system of claim 24, wherein theplatelet storage solution further comprises platelets contained therein.28. The system of claim 24, wherein the bag comprises side wallscomprising a film manufactured from a copolymer comprising ethylene andan acrylate.
 29. The system of claim 24, wherein the bag comprises sidewalls comprising a film manufactured from plasticized ultra highmolecular weight PVC.
 30. The system of claim 24, having >1.4×10⁹platelets/mL in the bag.
 31. The system of claim 14, further comprisinga leukocyte depletion filter assembly in fluid communication with thebag.
 32. A method of processing a biological fluid comprising: combininga platelet-containing biological fluid with the platelet resuspensionsolution of any one of claim 1, to provide a platelet- and plateletresuspension solution-containing fluid.
 33. The method of claim 32,including re-suspending the platelets in the platelet resuspensionsolution.
 34. The method of claim 33, including re-suspending theplatelets in the solution for a period of about 15 minutes to about 10hours.
 35. The method of claim 33, including re-suspending the plateletsin the solution for a period of about 30 minutes to about 6 hours. 36.The method of claim 32, further comprising adding a buffer to the fluidto provide a buffered platelet-containing fluid; and, storing thebuffered platelet-containing fluid in a gas permeable flexible bag forat least about 24 hours while maintaining the pH of the buffered fluid.37. The method of claim 36, wherein the buffered platelet-containingfluid is a leukocyte-depleted fluid.
 38. A method of processing abiological fluid comprising: obtaining a platelet-containing solutioncomprising leukocyte-depleted platelets and an aqueous solution having apH in the range of from about 6.6 to about 7.8; at least one buffer;dextrose; and citrate; wherein the solution is substantially free ofadenine; and storing the solution in a gas permeable flexible bag for atleast about 24 hours.
 39. A method of processing a biological fluidcomprising: combining a platelet-containing biological fluid with theplatelet storage solution of any claim 8 to provide a buffered platelet-and platelet storage solution-containing fluid.
 40. The method of claim39, wherein the buffered platelet- and platelet storagesolution-containing fluid is a leukocyte-depleted fluid.